The Research On Synthesis Of Novel Magnetic Nanocomposites And Their Application For Pullulanase Immobilization

Pullulanase(EC 3.2.1.41), which belong to the starch debranching enzymes, has became key enzymes in the starch processing industry. However, commercial pullulanase as a biocatalyst always presents some drawbacks that limit its popularization in food industry, such as easy deactivation at higher temperature, and inability of reutilization. Stability improvement of pullulanase and re-utility along with consecutive hydrolysis cycle can be achieved by immobilization of enzymes. Magnetic nanoparticles are usually considered to be ideal enzyme carriers. They have a large surface-area-volume ratio, allowing enzyme immobilization to be carried out under moderate conditions, thus retaining the catalytic activity of the enzyme. Furthermore, diffusion hindrance can be reduced because of the small size of the support. Therefore, they have been arousing increasing attention in field of enzyme immobilization. In this study, in situ minerization process for the preparation of a suitable support for the pullalanase immobilization through asorbtion, covalent lingking and encanpsulation were developed. With these methods, enzymes with high catalytic activity, high stability and easy separation from products were obtained. Moreover, the immobilized pullulanase were used for the preparation of resistant starch.Magnetic carrageenan nanoparticles were first prepared by in situ mineralization, then pullulanase were immobilized by the electrostatic interaction between the pullulanase/chitosan complex and Fe3O4-κ-carrageenan nanoparticles, and the as-prepared immobilized enzymes were characterized by VSM, TEM, FTIR, XRD, and TGA. The average particle size of the immobilized enzyme was 152 nm, and the magnetic property was improved as the charge of the sulfate groups was neutralized, Ms reached 42.6 emu/g. The activity retention of immobilized pullulanase and amount of enzyme loaded reached 95.5% and 96.3 mg/g, respectively, under optimal conditions. The immobilized enzyme exhibited great operational stability(retaining approximately 61% residual activity after ten consecutive reuses), demonstrating that enzyme leakage during the catalysis reaction was effciently reduced.The effect of chitosan molecular weight on the interaction between chitosan and pullulanase were comprehensively studied. The p Hc and p Hφ1 of complex was affected by CS Mw. Critical p H structure-forming event(p Hφ1) of the complex obtained with low molecular chitosan(50 k Da) shifted to lower p H compared to the other three CS-pullulanase types. Increase in chitosan molecular weight was associated with an increase in the surface charge and decrease in size distribution.The binding constants(Ksv)for the interaction between chitosan and pullulanase increase in the order CS-500 < CS-400 < CS-50< CS-200. Results of changes in secondary structures of pullulanase because of chitosan binding showed that the high binding affinity of chitosan-200 k Da to pullulanase induces more alterations of the protein secondary structure(decrease of α-helix from 31% to 19%), leading to the low enzyme activity. Pullulanase immobilized upon chitosan-50 complexation offered the most dramatic results of activity retention and stability; however, the pullulanase immobilized upon chitosan-200 complexation exhibited the worst results.The CS-MNPs were synthesized by in situ mineralization of Fe3O4 in chitosan hydrogel and used as support for pullulanase immobilization. The CS-MNPs were characterized by TEM, FTIR, XRD and TGA. As the chelation effect confines the iron ions diffusion and crystal growth of magnetite, the nanoparticles have a narrow size distribution of 5 nm. The content of chitosan and Fe3O4 was 12.7% and 83.28%, respectively. In process of immobilization, enzymatic activity was affected greatly by the enzyme concentration and p H. The high activity retention was obtained at optimal binding enzyme concentration of 60 μg/ml and binding p H(3.5). Compared to free pullulanase, some enzymatic characteristics were improved after immobilization, such as higher p H and thermal stability, however, the optimal p H(3.5) of immobilized pullulanase shifted to more acid side.Factors relating to different immobilization ways(EDC or GA crosslinking, Na BH4 reduction), covalent density and arm length on the immobilization were studied. More catalytic activity was kept by covalent attachment formed using GA than formed using EDC. Since immobilization is based on the reaction of lysine-amino groups, the further reduction of Schiff base by Na BH4 yield more stable secondary amino bonds and therefore the best operational stability. However, there would be a possible reduction of disulfide bonds of the enzyme simultaneously, resulting in the slight decrease of activity after reduction. Results showed that the maximal activity retention of immobilized pullulanase(86%) was observed at a surface amino density of 1.6 per nm2, corresponding to 57.6 per 3600? ?2 covalant bonds that one pullulanase molecule can form with magnetic chitosan/Fe3O4 nanoparticle, approximately. The longer spacer arm(15%, dimer) gave the higher activity retention(89%) than the shorter spacer arm(0.5%, monomer) gave.Based on Fe3O4-chitosan nanoparticles, immobilized pullulanase of magnetic chitosan nanoparticles as core with polymer shell were synthesized using sol-gel method, and characterized by VSM, TEM, FTIR and TGA. In the sol-gel reaction, morphology of gel particle obtained after the addition of enzyme is more structured, and the particle showed a better magnetic property(Ms 26.8 emu/g); the content of pullulanase is about 8.83%; optimum immobilization conditions: the proportion of silane precursors of OTES/TEOS = 1/2, enzyme concentration of 0.484 mg/ml sol. Cross-linking was further brought on the basis of the conventional sol-gel encapsulation. Results showed that immobilized enzymes obtained through chemically crosslinking combined with sol-gel encapsulation showed significantly higher stability and reusability than the immobilized enzymes through sole sol-gel encapsulation, indicating the improvement of enzyme leakage. Considering both the stability and activity retention, the enzyme immobilized through carbodiimide activation and cross-linking with the magnetic carrier followed by the sol-gel encapsulation showed better performance than the other two immobilized enzymes, which showed 81.3% activity retention, and retained 87% of original enzyme activity after 6 times repeated uses.The three aforesaid synthesized immobilized enzymes through adsorption, covalent or encapsulation were used to prepare resistant starch using normal maize starch. Maize starch treated with autoclave yield 12.5% RS, maize starch treated with autoclave and free pullulanase debranching yield 35.1% RS, while maize starch treated with autoclave and immobilized pullulanase(by encapsulation) debranching yield the maximum RS content(43.3%).can reach. Seen from the results of liquid chromatography, the content of low molecular weight linear oligosaccharides of starch treated with immobilized enzyme is higher than that of starch treated with free enzyme, and a characteristic peak corresponding molecular weight of about 6200 appeared. DSC results showed that the peak temperature of starch treated with enzyme debranching shifted to higher side(130 °C). After 8 repeated uses, starch debranched by immobilized enzymes through adsorption, covalent or encapsulation yield 61.3%, 70.4% and 75.7% of original RS content, respectively, indicating that the use of immobilized enzymes prepared by encapsulation showed better performance in resistant starch production than the other two immobilized enzymes.